Insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II), previously known as somatomedins, are structurally related to insulin and are the two most abundant polypeptide growth factors that circulate in human plasma (1). IGFs are multipotent growth factors responsible for normal tissue growth and regeneration. In addition, IGFs have been suggested to have beneficial effects on glucose homeostasis by its glucose lowering and insulin sensitizing actions. However, not all effects of IGFs are considered to be favorable; thus, epidemiological studies suggest that IGF-I and IGF-II are also involved in the development of common cancers, atherosclerosis and type 2 diabetes (2).
The IGFs are secreted by a number of cell types, including smooth muscle cells and macrophages. The cellular effects of the IGFs are mediated by membrane bound high affinity receptors. IGF receptors are of two distinct types and are expressed by a wide variety of cells. The IGFs are potent smooth muscle cell mitogens and it was suggested that these polypeptides contribute to the formation of the atherosclerotic lesion by paracrine, autocrine or endocrine mechanisms (3).
In plasma and other biological fluids, IGFs are complexed with proteins from the family of structurally related proteins, called IGF-binding proteins (IGFBPs). IGFBPs bind approximately 99% of the circulating IGF pool. This family of proteins includes at least six IGFBPs. IGFBPs are distinct from the IGF receptors. It is thought that IGFBPs modulate the effects of the IGFs in different tissues, including bone (4).
IGFBP-4 was initially purified from human bone cell conditioned medium as a 25 kDa protein. Later, IGFBP-4 was also purified from conditioned medium collected from a variety of cell types, and the expression of IGFBP-4 was revealed in various cell types (5, 6). The primary structure of the human IGFBP-4 protein was deduced from human placenta and osteosarcoma complementary DNA (cDNA) libraries (7, 8). The cDNA for human IGFBP-4 encodes a 258-residue protein that is processed, by removal of the signal sequence, to a mature protein of 237 residues (25.6 kDa) with a single asparagine-linked glycosylation site (7). Although various cell types when in culture secrete both glycosylated (28-29 kDa) and nonglycosylated (24-25 kDa) forms of IGFBP-4, the nonglycosylated is typically the most abundant in normal human blood (5, 6).
IGFBP-4 is unique among the six IGFBPs in having two extra cysteine residues in the variable L-domain. These unique properties of IGFBP-4 may be responsible for the distinctive biological functions of IGFBP-4 (13).
Mean level of IGFBP-4 in adult human serum is higher than those of IGFBP-1, IGFBP-2, and IGFBP-6; is similar to that of IGFBP-5; and is less than IGFBP-3 level. The trend of serum IGFBP-4 to increase with age is similar to that reported for IGFBP-1 and IGFBP-2, but is different from the declining with age levels of IGFBP-3 and IGFBP-5, suggesting that in human serum levels of the different IGFBPs are differentially regulated with advancing age (12).
Although the exact functional role for serum IGFBP-4 is not absolutely clear, in vitro studies have shown that IGFBP-4 inhibits IGF activity in bone cells and other cell types. Mohan et al. (9, 10) demonstrated that IGFBP-4 inhibited both IGF-I- and IGF-II-induced cell proliferation of embryonic chick calvaria cells and MC3T3-E1 mouse osteoblasts. IGFBP-4 inhibits IGF-I- and IGF-II stimulated the DNA synthesis in a variety of cell types (6).
IGFBP-4 synthesis may be regulated by systemic hormones and local growth factors at the transcriptional or posttranscriptional level (11). Studies in vitro revealed that parathyroid hormone, 1,25-dihydroxyvitamin D, IGF-I, IGF-II, transforming growth factor-beta, and osteogenic protein-1/bone morphogenetic protein-7 are major regulators of IGFBP-4 production in human bone cells (8,9).
Specific proteolysis is a major regulatory mechanism of IGFBP-4 functions. An IGF-dependent IGFBP-4-specific protease was first reported in the media conditioned by both human and sheep dermal fibroblasts. This protease was later identified as pregnancy-associated plasma protein-A (PAPP-A). The same proteolytic activity was also detected in the conditioned media from cultivated human osteoblasts, vascular smooth muscle cells, granulose cells, trophoblast and decidualized endometrial stromal cells, as well as in ovarian follicular fluid and human pregnancy serum (13).
PAPP-A was first isolated from human pregnancy serum in 1974 as a tetrameric complex with proform of eosinophil major basic protein (proMBP). It was revealed that PAPP-A belongs to the large metzincin family of metalloproteases. It was shown that recombinant PAPP-A is an active protease able to cleave IGFBP-4 at a single site, between M135/K136 (one letter amino acid residues code is used). IGFBP-4 cleavage by PAPP-A is possible only in case when IGFBP is complexed with IGF. PAPP-A also cleaves IGFBP-5 between S143/K144, but in this case the presence of IGF is not required.
Pregnancy associated plasma protein A was first suggested as a biological marker of atherosclerotic plaques instability after a study by Bayes-Genis et al. (16). These authors have demonstrated high levels of PAPP-A in the extracellular matrix of unstable plaques. Several studies have shown that concentration of PAPP-A in blood of patients with acute coronary syndrome (ACS) is higher than in blood of patients with stable coronary artery disease or control subjects. Thus PAPP-A was suggested as a marker of cardiovascular diseases associated with coronary artery blood clotting, such as unstable angina and myocardial infarction.
It was hypothesized that in atherosclerotic plaques PAPP-A expressed by activated smooth muscles cells could function as an active enzyme cleaving IGFBP-4 complexed with IGF, thus enhancing IGF bioavailability. The IGF system might contribute to the atherosclerotic plaque development, destabilization, and rupture leading to acute coronary events (17).
It was shown that IGFBP-4 is expressed by different cells of tumor origin, such as lung adenocarcinoma, non-small-cell lung cancer, breast cancer, colon carcinoma, follicular thyroid carcinoma, gastric cancer, glioma, hepatoma, myeloma, neuroblastoma, osteosarcoma and prostate cancer. In vitro and in vivo studies suggest that IGFBP-4 plays an important role in the growth regulation of a variety of tumors, possibly by inhibiting autocrine IGF actions. Regulation of IGF bioavailability may play crucial role in tumor growth and development (13).
Available evidences seem to suggest that plasma measurements of PAPP-A concentration could be valuable for the discrimination of patients with unstable atherosclerotic plaques as well as for identification of patients with cancer. However, it has now turned out that precise PAPP-A blood measurements are not an easy task. This is, first of all, due to the fact that there are extremely low levels of this protein in patients' plasma. It was also shown that heparin injections could also influence the PAPP-A levels in patients' plasma (18).
We have suggested that the measurements of the products of PAPP-A enzyme activity could be of higher clinical value than direct PAPP-A measurements because such products should be presented in blood in higher concentrations than PAPP-A and their concentration in blood should not be affected by heparin injections.
The present invention is devoted to the utilization of IGFBP-4 fragments as markers of increased PAPP-A activity in the body, and consequently, as markers of the different diseases associated with increased PAPP-A concentrations and activity. The immunoassay methods designed for specific measurements of proteolytic fragments of IGFBP-4 regardless of the presence of intact IGFBP-4 in patients' samples could be of practical value for the diagnosis or prediction of various pathologies including ACS and cancer.